An FT-IR Study on Packing Defects in Mixed β-Aggregates of Poly(l-glutamic acid) and Poly(d-glutamic acid): A High-Pressure Rescue from a Kinetic Trap

Abstract

Under favorable conditions of pH and temperature, poly(L-glutamic acid) (PLGA) adopts different types of secondary and quaternary structures, which include spiral assemblies of amyloid-like fibrils. Heating of acidified solutions of PLGA (or PDGA) triggers formation of β(2)-type aggregates with morphological and tinctorial properties typical for amyloid fibrils. In contrast to regular antiparallel β-sheet (β(1)), the amide I' vibrational band of β(2)-fibrils is unusually red-shifted below 1600 cm(-1), which has been attributed to bifurcated hydrogen bonds coupling C═O and N-D groups of the main chains to glutamic acid side chains. However, unlike for pure PLGA, the amide I' band of aggregates precipitating from racemic mixtures of PLGA and PDGA (β(1)) is dominated by components at 1613 and 1685 cm(-1)-typically associated with intermolecular antiparallel β-sheets. The coaggregation of PLGA and PDGA chains is slower and biphasic and leads to less-structured assemblies of fibrils, which is reflected in scanning electron microscopy images, sedimentation properties, and fluorescence intensity after staining with thioflavin T. The β(1)-type aggregates are metastable, and they slowly convert to fibrils with the infrared characteristics of β(2)-type fibrils. The process is dramatically accelerated under high pressure. This implies the presence of void volumes within structural defects in racemic aggregates, preventing the precise alignment of main and side chains necessary to zip up ladders of bifurcated hydrogen bonds. As thermodynamic costs associated with maintaining void volumes within the racemic aggregate increase under high pressure, a hyperbaric treatment of misaligned chains leads to rectifying the packing defects and formation of the more compact form of fibrils.

Covalent Defects Restrict Supramolecular Self-Assembly of Homopolypeptides: Case Study of β2-Fibrils of Poly-L-Glutamic Acid

“…The spectral data presented in Figure 2B strongly supports the idea that β 2 -fibrils are the proper end product of defect-free amyloidogenic self-assembly of PLGA chains [20]–[22]. Formation of fibrils with β 2 -type infrared features requires the occurrence of additional stabilizing interactions: bifurcated hydrogen bonds involving Glu carboxyl groups and main chain carbonyl groups.…”

“…Formation of fibrils with β 2 -type infrared features requires the occurrence of additional stabilizing interactions: bifurcated hydrogen bonds involving Glu carboxyl groups and main chain carbonyl groups. This may explain the earlier observations that in mixtures of PLGA and PDGA chains, β 2 -fibrils appear to be more thermodynamically stable than β 1 -aggregates [22]. Certainly, the dense packing and strict steric requirements for three-center hydrogen bonds narrowly define the structure of β 2 -fibrils.…”

“…Certainly, the dense packing and strict steric requirements for three-center hydrogen bonds narrowly define the structure of β 2 -fibrils. On the other hand, aggregates with the β 1 -like spectral features are more likely to constitute a broad ensemble of agglomerates unable to establish such bonding patterns for one reason (kinetic traps caused by co-aggregation of PLGA and PDGA chains [22]) or another (e.g. random covalent modifications of side chains used in this study).…”

“…In all our previous investigations employing high quality linear PLGA chains [20]–[21] only β 2 -aggregates were observed, although the aggregation conditions were essentially the same or very similar to those used by Fändrich and Dobson. In our earlier studies on acid-and-temperature-induced conformational transitions in PLGA, β 1 -aggregates were detected only as metastable transient forms occurring upon co–aggregation of PLGA and PDGA (poly-D-glutamic acid) [22]. This has led us to speculate that the amyloidogenic self-assembly of poly-Glu chains unperturbed by defects, or by polydispersity of the polymer, should produce higher order structures with β 2 -like infrared characteristics.…”

Temperature dependence of amino acid side chain IR absorptions in the amide I' region

“…1,2 While inherently low resolution, the IR has proven very useful for monitoring structural changes due to protein fol-ding=unfolding, [3][4][5] aggregation, [6][7][8][9] protein interactions, 10 and enzymatic reactions. 1,2 While inherently low resolution, the IR has proven very useful for monitoring structural changes due to protein fol-ding=unfolding, [3][4][5] aggregation, [6][7][8][9] protein interactions, 10 and enzymatic reactions.…”